The binding of epidermal growth factor (EGF), a 53-residue polypeptide hormone, to its plasma membrane receptor initiates a broad array of cellular responses, ultimately culminating in mitosis. The receptor is a transmembrane glycoprotein of Mr = 170,000, the sequence of which has been deduced from the cDNA for the receptor. When EGF binds to the extracytoplasmic domain of the receptor, (1) a Tyr-specific protein kinase within of the cytoplasmic domain of the receptor is activated, and (2) the receptor dimerizes. The cause-and-effect relationship of these two events if not yet well understood. A critical residue, Lys721, in the active site of the kinase domain has been identified by affinity labeling and protein microsequencing. Site-directed mutagenesis of Lys721 to Met or Ala abolishes the kinase activity of the receptor. Further, when these mutant receptors are expressed in cells that do not ordinarily bear EGF receptors, EGF does not evoke any of the usual cellular responses, strongly suggesting that the activation of the kinase activity by the binding of EGF is the critical event in transmembrane signaling. The long term goal of this project is to develop an in-depth understanding of the protein chemistry and enzymology of the EGF receptor as it relates to the mechanism of signal transduction. This proposal addresses this long term goal through six specific aims: (1) In order to identify residues of the receptor immediately adjacent to bound EGF, site-directed mutants of murine EGF will be prepared, each containing one specific residue mutated to Cys. (2) The mutant EGFs will be affinity cross-linked to the receptor with thiol-specific, kinetically driven cross-linking reagent, and the sites of cross-linking will be identified by protein microsequencing. (3) The same Cys mutants will be doubly modified with amino- and thiol-specific, kinetically driven cross- linkers to test whether one or both receptor monomers contribute to the EGF binding site. (4) Residues near the substrate recognition site will be identified. Peptide substrates will be affinity cross-linked to the receptor, or a photoreactive amino acid will be incorporated into peptide substrates that will be used to photoaffinity label the peptide substrate site. (5) A recently identified noncompetitive peptide inhibitory site will be mapped by an approach parallel to that used for the substrate binding site. (6) A new ATP affinity label in which the ribose ring is modified with a reactive group will be synthesized and tested as an affinity label for the ATP site. Residues modified by these methods will be identified by protein microsequencing.